The members of the genus Bordetella are pathogenic microorganisms involved in the infection of the respiratory tract. The genus is comprised of four species; B. pertussis, B. parapertussis, B. bronchiseptica, and B. avium. The most virulent species to man is B. pertussis, which is the etiologic agent of whooping cough.
Current conventional pertussis vaccines contain whole but inactivated B. pertussis cells. Such cells are inactivated by treatment at 56.degree. C. for 30 minutes and/or treatment with formaldehyde. In spite of inactivation, such whole cell vaccines retain a substantial amount of toxicity.
As a result, alternate pertussis vaccines are available which are prepared from avirulent or toxin-deficient strains of B. pertussis. However, these vaccines have proven to be much less protective than those prepared from virulent strains. See, for example, Wardlaw et al., J Med Microbiol 9:89-100 (1976).
B. pertussis produces a number of toxins (pertussis toxin, adenylate cyclase, dermonecrotic toxin, and trachael cytotoxin) which destroy the clearance mechanisms of the respiratory tract, or interfere with the immune response (F. Mooi, Antonie van Leeuwenhoek, 54:465-474 (1988)). A wide variety of biological activities, such as histamine sensitization, insulin secretion, lymphocytosis promotion and immunopotentiating effects can be attributed to the pertussis toxin (J. Munoz, in Pertussis Toxin, p. 1-18, Sekura et al., Eds., Academic Press, New York, 1985). In addition, it has been shown that the administration of the B. pertussis toxin in mice protected them against subsequent challenge (Munoz et al., Infect Immun 32:243-250 (1981)). Pertussis toxin is therefore an important constituent in a vaccine against whooping cough and is included in the acellular component vaccines being tested and used in several countries (Sato et al., Lancet, 1984-I:122 (1984)). Paradoxically, the pertussis toxin, which is capable of eliciting an immune response, may itself be responsible for the harmful side effects associated with current vaccines (Steinman et al., Proc. Natl Acad Sci USA, 82:8733 (1985)). These harmful effects can range from simple flushing to permanent neurological damage and in some instances, death.
The pertussis toxin is composed of five different subunits, designated S1 to S5 based on their electrophoretic migration in SDS-polyacrylamide gels. The subunits associate in the molar ratio of 1:1:1:2:1, respectively, to form the holotoxin. Functionally, the pertussis toxin can be divided into the A protomer, or S1 subunit, which contains adenosine diphosphate (ADP)-ribosylation activity, and the B-oligomer, comprised of subunits S2 through S5, which contains target cell receptor binding activities. Thus the B-oligomer is essential in bringing the A protomer into contact with the target-cell's membrane.
Locht et al., Science 232: 1258-64 (1986), disclose that the subunits of the pertussis toxin are encoded by closely linked cistrons. Locht et al. further disclose the nucleotide sequence of the B. pertussis toxin gene and the amino acid sequences for the individual subunits.
Locht et al., NAR 14:3251-61 (1986), reveal the cloning of a 4.5 kb DNA fragment from the B. pertussis toxin gene containing at least the S4 subunit and a portion of another subunit gene. Sequence analysis revealed that the mature S4 subunit is derived by proteolytic cleavage of a precursor molecule.
Nicosia et al., Infect Immun 55:963-7 (1987), disclose expression of each of the five B. pertussis toxin subunits as fusions to DNA polymerase MS2. Antisera raised to these proteins were found not to be immunoprotective in vivo or in vitro.
Locht et al., Infect Immun 55:2546-2553 (1987), disclose the expression of the S1 and S2 subunits of pertussis toxin in E. coli as fusions to 6 amino acid residues of beta-gacactosidase followed by 5 amino acids encoded by a polylinker. It was disclosed that the recombinant S1 subunit displayed enzymatic activities. A truncated version of the S1 subunit was disclosed in which the last 48 amino acid residues, i.e., the carboxy terminus, was deleted.
Sclavo SpA, EP-A-232,229, published Aug. 12, 1987, disclose the cloning and expression of a B. Pertussis toxin gene, which contains subunits S1 through S5 in E. coli.
Bellini et al., EP-A-281,530, published Sep. 7, 1988, disclose expression of mature B. pertussis subunits in B. subtilis
Burnette et al., EP-A-306,318, published Mar. 8, 1989, report the subcloning and expression of individual B. pertussis toxin subunits in E. coli. Burnette et al. disclose that the S4 subunit could only be expressed upon removal of the signal peptide coding sequence. Burnette et al. also disclose S1 subunit analogs expressed in E. coli with modifications between amino acids Val.sup.7 to Pro.sup.14.
Burns et al., U.S. Pat. No. 4,845,036, disclose a method for isolating the wild-type B. pertussis B-oligomer (i.e., subunits S2-S5) by dissociation of the holotoxin (i.e., subunits S1-S5).
Sato et al., EP-A-296,765, published Dec. 28, 1988, disclose B. pertussis variants which produce mutant pertussis toxin proteins. The variants arose from exposure of virulent B. pertussis with nitrosoguanidine, a known mutagen.
M. Ui, (in Pathogenesis and Immunity in Pertussis, Wardlaw et al., eds., p.121-145, Wiley & Sons, Chichester, 1988) discloses that certain chemical modifications, e.g., acylation, of the pertussis toxin lysine residues eliminate all biological activity. Methylation of the pertussis toxin, which also modifies lysine residues, does not affect the ADP-ribosylation activity but does reduce or abolish certain biological activities associated with the B-oligomer, for example, mitogenic activity, stimulation of glucose oxidation, promotion of lymphocytosis and histamine-sensitizing activity. Ui further discloses that methylation of dimer D2 (i.e., pertussis toxin subunits S3-S4), but not dimer D1 (i.e., pertussis toxin subunits S2-S4) or subunit S5, eliminates the mitogenic activity associated with the B-oligomer. There is no disclosure or suggestion, however, as to which specific regions or specific lysine residues of the B-oligomer are involved in the methylation or acylation.
Hausman et al., Infect Immun 57:1760-64 (1989), disclose immunization of mice with the pertussis toxin dimeric subunits, D1 (i.e., S2-S4) and D2 (i.e., S3-S4). The antisera raised to these dimers were able to recognize B. pertussis toxin and neutralize its toxic effects in vitro.
Capiau et al., U.S. Patent application Ser. No. 07/222,991,*filed Jul. 22, 1988 disclose modification of the B. pertussis toxin S1 subunit at amino acid position 26 (i.e., tryptophan). This residue can be modified either chemically or by site-directed mutagenesis to substantially inactivate the enzymatic activity of the S1 subunit.
Bellini et al., Gene, 69:325-330 (1988), recite a general method for site-directed mutagenesis for double-stranded plasmid DNA. Bellini et al. disclose that their method is particularly valuable where long deletions are needed. Exemplified is the deletion of the B. pertussis S2 subunit signal sequence coding region located on an E. coli--B. subtilis shuttle vector.
Black et al., EP-A-275, 689, published Jul., 27, 1988 and Infect Immun 55:2465-70 (1987), disclose expression of the S4 subunit in E. coli. In addition, Black et al. disclose mutations in the B. pertussis toxin gene that were either deletions generated by Bal31 exonuclease or insertions with the kanamycin resistance gene. These mutations were then introduced by allelic exchange into the B. pertussis chromosome.
Klein et al., EP-A-322,115, published Jun. 28, 1989, disclose substitution mutations of the B. pertussis toxin. Klein et al. also disclose deletion mutations of the S1 subunit. However, of the deletion mutations disclosed, only one mutation, Glu.sup.129, was weakly reactive against antibodies to the S1 subunit.
It is an object of this invention to provide an improved B. pertussis vaccine comprising a modified B. pertussis toxin or subunits thereof which are immunogenic, yet non-toxic.